Fungi occurrence on seeds of field pea

J.: Fungi occurrence on 77–89, 2008. Seeds of four edible cultivars of Pisum sativum and three fodder harvested in 2004-2006 from eight localities, scattered in all region suitable for pea production in Poland, were evaluated for fungi occurrence on CN agar medium in Petri plates. The highest number (27) of species was isolated in 2004, while the lowest (16) in 2006. Number of fungi inhabiting seeds was influenced mainly by environmental conditions of locality and years. Alternaria alternata dominated in each sample of 450 seeds. Species of Penicillium contaminated seeds as the next and infection by Stemphylium botryosum was at similar level . Fusarium poae was the most often occurring species of this genera. Pea specific pathogens: Mycosphaerella pinodes, Phoma pinodella and Ascochyta pisi infected more seeds in 2004 and 2005 than 2006, and at the last season only A. pisi was noted. In general, level of infection by those pathogens was low, reaching on an average only 2.56%, with the highest for A. pisi , and the lowest for M. pinodes.

Fungi inhabiting pea seeds in Poland were already described, but on genotypes issued earlier. Now the increasing importance of dry pea seed production for edible ACTA MYCOLOGICA Vol. 43 (1): 77-89 2008 and animal feeding (fodder) purposes caused breeders interest in releasing new cultivars of field pea. Seeds of recently introduced cultivars were the objective for evaluation of any fungi occurring on them.

MATERIALS AND METHODS
Seeds of four edible and three fodder cultivars, six bred in Poland and one in Czech, registered between 1995-2006, were tested for fungi occurrence (Tab. 1). Seeds for evaluation were harvested in 2004, 2005 and 2006 from plants cultivated in fields of 14 localities situated in seven different regions of Poland, suitable for pea production. Cultivars of each type were planted in eight localities, six different and two the same, since at Cicibór (C) and Kawęczyn (KA) were tested edible cultivars requiring better soils for cultivation and also fodder one of light soil requirements.
A sample of 450 seeds of each cultivar, collected at 8 localities were studied every year. Surface sterilized seeds (Marcinkowska, Boros, in print) were placed on Coon's (CN) agar medium into a Petri plate (Pp) 10 cm in diameter. A plate contained 15 seeds. Evaluation was done in two equal series (15 Pp x 15 seeds equals 225), first started in late December, second by the end of February. The same incubation conditions were provided for seeds of both series (Marcinkowska 1997). Identification of fungi was done following different keys (Marcinkowska 1998;2003). List of fungi occurred on seeds was given in table 2. Data were taken on the eight day since plating.
Number of identified species was counted together for both series and changed for percent of a sample. For statistical evaluation percentage data were transformed according to Bliss (TP) and used in Statgraphics Plus programme. Majority of occurring fungi (Tabs 3, 4), that means nine common species and 4 genera of more than one species (Aspergillus, Cladosporium, Fusarium, Penicillium), were covered by statistical analyses. Statistical analysis were also done for comparison of frequency of three species responsible for ascochyta blight and Alternaria alternata on tested seeds Ta b l e 1 Names of cultivars and origin of tested seeds  (Tabs 5,6). Incidence of fungi on seeds was evaluated according to Fisher's least significant difference (LSD) procedure, the method used to discriminate among the means at the 95% confidence level.

RESULTS
On evaluated seeds of 7 cultivars, 4 edible and 3 fodder, 27 species of fungi were identified in 2004, 22 in 2005and 16 in 2006.
Statistical analysis of data concern majority of species occurring on seeds indicated that number of fungi inhabiting edible cultivars and fodder ones depended significantly on their species and year (Tab. 3). Significance of differences between locality and cultivar was various for both groups of cultivars.
The most often isolated fungus from seeds, over cultivars, localities and years, was A. alternata, as often as Penicillium spp. from fodder cultivars, but for edible one the next was S. botryosum and Penicillium spp., both of homogenous group, of lower intensity, to which partly belonged also A. pisi (Tab. 4 When statistical analysis was done only for A. alternata, the species most often inhabiting seeds, and the specific pea pathogens, M. pinodes (syn. Didymella pinodes (Berk. et Blox.) Petrak) anamorph Ascochyta pinodes L. K. Jones), A. pisi and P. pinodella, significant differences were found again for fungi occurring on seeds of both cultivar groups, and for edible -on localities, but for fodder -in years (Tab. 5).
A. alternata dominated, reaching respectively 10.29 and 8.33 percent, on edible and fodder cultivar seeds compared to species responsible for ascochyta blight, which were covered by second homogenous group (Tab. 6).
Localities influenced also seed contamination but statistically proved differences were noted only for edible cultivars both when majority of fungi and only Ta b l e 3 Analysis of variance for seed contamination of edible and fodder cultivars by majority of fungi species occurring over cultivars, localities and years When inhabitance of cultivars over fungi, localities and years was analyzed statistically documented differences were only reported for fodder one (Tab. 3). Sokolik and Zagłoba were less contaminated by all fungi compare to Hubal (Tab. 9).
There were no significant differences between occurrence each of ascochyta blight fungi on seeds of both types of cultivars independently on cultivar, year and locality (Tab.6) but when separately the factors were considered Terno showed the highest infection in 3 out of 8 localities, reaching respectively, 20.22 percent in Cici-  bór, 19.11 in Krzyżewo and 12.4 in Radostowo (Tab. 10). Data were given as a sum of seed percent infected by M. pinodes, A. pisi and P. pinodella. The second stronger infected was Set in Krzyżewo (17.56) and Radostowo (5.11). Such high infection was only observed on seeds harvested in 2004, but those of 2006 were almost clean, since only from single ones of Terno, Set and Ramrod fungi responsible for ascochyta blight were isolated. During three years no infection was noted on seeds from Chrząstowo and Sulejów, but the highest was found at Krzyżewo, Radostowo, Cicibór and Rarwino. In the period of studies (2004)(2005)(2006) seed infection by fungi responsible for ascochyta blight was very low (Fig. 1). A. pisi was isolated most often, independently on cultivars, years and localities totally 1.54 % of seeds, from 1.46 % in 2004 to 0.02 % in 2006 were infected. This species was the only one noted on seeds harvested in 2006. It dominated on seeds harvested in 2004, but in the next year P. pinodella was the most often isolated species.

DISCUSSION
Occurrence frequency of fungi inhabiting pea seeds was first of all depended on species. Seeds of all sample transmitted A. alternata and this species dominated among other fungi. The presented results supported the earlier obtained for field pea by Filipowicz (1976) and Marcinkowska (1997), green pea (Czyżewska 1976) and dry edible pea (Marcinkowska 1998). The next in this study were species of Penicillium with P. expansum, occurring in majority, but also P. claviforme contaminated many seeds. Filipowicz (1976) reported even more often Penicillium sp. than A. tenuis Nees (syn. A. alternata). Also Marcinkowska (1998) noted in 1992 common occurrence of Penicillium sp. Frequency of Stemphylium botryosum, weak polyphagous pathogen, the third more often inhabiting species, was similar to reported earlier (Grzelak, Iłłakowicz 1973;Marcinkowska 1997Marcinkowska , 1998. It was important to notice that seeds were contaminated not only by Penicillium spp. but also Aspergillus spp., of which genera some species, like A. flavus and so P. expansum could be able to produce mycotoxins (Kozakiewicz 1990(Kozakiewicz , 1992. So one had to realize the presence of those species on seeds as dangerous, but on the other hand not all isolates, even of harmful species could produce mycotoxins, and their amount might be also changeable depend on different factors, like temperature or light of environment. Number of isolated species differed for various studies, Filipowicz (1976) reported the highest (30), the lowest (8) Grzelak and Iłłakowicz (1973), but in presented work were isolated 27, in 2004 -slightly cooler and of higher rainfalls, and 16 in 2006 of wormer and drier vegetation season (Figs 4, 5). Not only the number but also composition of species inhabiting seeds was influenced by weather conditions, particularly pea pathogenic fungi for which development a drop of water is necessary to infect a plant. Specific pathogens of peas, ascochyta complex fungi, were noted each season less frequently to common saprobe, A. alternata, like it happened for dry seeds in early 90.ties (Marcinkowska 1998). While on fall-planted Austrian winter pea, even A. alternata occurred very often, P. pinodella dominated in Poland on seeds in 1994 and M. pinodes in 1993 (Marcinkowska 1997), like in Canada (Morrall et al. 2005) and France in recent years (Fougereux et al. 2006). In Poland also Grzelak and Iłłakowicz (1973) and Filipowicz (1976) reported common occurrence of fungi responsible for ascochyta blight, but with the highest for A. pisi. These earlier results concern the last species frequency were supported by the recent data from Canada (Morrall et al. 2005) and the presented one, even level of seeds infected by ascochyta complex fungi was very low. A. pisi, took first place and was the only one out of three, however very sporadic, in the warmest growing season of 2006. On the other hand no significant differences were found between frequency of M. pinodes, P. pinodella and A. pisi, when they were compared to A. alternata. The level of seed infection by M. pinodes and P. pinodella in case of majority of species inhabiting was similar for both groups of cultivars, and lower comparing to A. pisi. The last species infected significantly more seeds of edible cultivars to fodder one. Even more to F. poae, the saprobic species of Fusarium inhabiting pea seeds most often. This domination was already proved by Filipowicz (1976) and Marcinkowska (1993Marcinkowska ( , 1997Marcinkowska ( , 1998. The number of isolated Fusarium species was lower to obtained by Filipowicz (1976) but the same as Marcinkowska (1993Marcinkowska ( , 1997, however the prevalence of species varied between reports. Among pathogenic species Czyżewska (1976) and Filipowicz (1976) noted more often F. oxysporum. In this study was also found F. solani reported earlier on seeds of winter pea (Marcinkowska 1997).
Variability of mycobiota was influenced, besides fungal species and weather conditions of the growing season, also by characters of cultivars and environmental conditions of localities. The last factor was combined with local weather or even microclimate of field where peas were growing in different years. Particularly rainfalls increased frequency of fungi inhabiting seeds, so for locality characterized by higher precipitation and usually lower temperatures (Figs. 4 and 5) many more fungi were isolated. The influence of weather was also reported from other studies (Bathgate et al. 1989;Xue et al. 1997;Roger et al. 1999;Marcinkowska 1998Marcinkowska , 2002Morrall et al. 2005;Fougereux et al. 2006). Presented data supported positive (2005 and 2004) or negative (2006) influence of weather on seed infection by plant pathogens but also their inhabitance by saprobic species, especially on cultivars suitable for light soil.
Influence of cultivars on fungal species occurrence was proved by several authors (Filipowicz 1993;Fougeroux et al. 2006;Marcinkowska 1997Marcinkowska , 1998Marcinkowska , 2002Xue et al. 1996). These data reported only such dependence considered all fungi on fodder cultivars. The obtained results supported the earlier reports cited here, done by different researches in various countries, that incidence of ascochyta blight fungi was influenced by many factors, changeable in between studies. According to Fougereux et al. (2006) and Morrall et al. (2006) these were mainly years, production area (localities) and type of crop (winter or spring pea). CONCLUSIONS 1. Many more seeds were inhabited by saprobic fungi than plant pathogenic. Number of fungi occurring on edible and fodder cultivars depended mainly on fungal species and year.
2. Alternaria alternate was the most often occurring species on both types of cultivars, next was Penicillium spp.
3. Stemphylium botryosum, the weak pathogen, was isolated most often from seeds of both cultivar types compare to all other pathogenic fungi.
4. Fungi responsible for ascochyta blight infected seeds sporadically. They were recovered with various intensity from seeds of both types of cultivars. Ascochyta pisi was most often isolated. 5. No clear response of tested pea cultivars to ascochyta blight fungi occurrence on their seeds was found since hostplant reaction was strongly influenced by environmental conditions (locality and year). 6. Differences in seed contamination by any fungi species were only found for fodder cultivars.